白藜芦醇对人肝癌HepG2细胞增殖及自噬作用的影响

Effect of resveratrol on proliferation and autophagy of human liver cancer HepG2 cells

目的观察白藜芦醇(resveratrol,Res)对人肝癌细胞Hep G2增殖及自噬作用的影响,探讨其可能的抗肿瘤机制。方法用不同浓度的Res作用Hep G2细胞24、48和72 h后,采用CCK-8法检测Res对细胞增殖的影响,丹酰尸胺(monodancyldadverine,MDC)染色法检测Res对Hep G2细胞自噬体形成的影响;用20μmol/L Res和20μmol/L Res+5 mmol/L 3-甲基腺嘌呤(3-methyladenine,3-MA)分别作用Hep G2细胞24 h,Western blot法检测自噬相关基因Beclin 1、LC3和P62的表达情况,同时检测PI3K/Akt/m TOR通路中,PI3K、p-Akt(Akt)和p-m TOR(m ROR)的表达情况。结果 Res作用Hep G2细胞后,能够明显抑制细胞增殖,呈剂量-时间依赖性;可促进Hep G2细胞内自噬体的形成。Western blot结果显示,Res作用Hep G2细胞后,可提高LC3Ⅱ/Ⅰ的比值,促进Beclin 1的表达,降低作用底物P62的表达,而3-MA作用细胞后,LC3Ⅱ/LC3Ⅰ比值、Beclin 1的表达水平均下降,P62的表达水平上调;Res还可显著降低细胞中PI3K、p-Akt和p-m TOR的表达。结论 Res可诱导自噬抑制Hep G2细胞的增殖,其机制可能与Res抑制PI3K/Akt/m TOR通路,进而上调自噬相关Beclin 1和LC3的表达以及下调P62的表达有关。

Objective To observe the effect of resveratrol（Res） on proliferation and autophagy of human liver cancer Hep G2 cells and investigate the possible anti-tumor mechanism of Res. Methods Hep G2 cells were treated with Res at various concentrations for 24, 48 and 72 h, determined for proliferation level by CCK-8 assay, and observed for formation of autophagosome by monodansylcadaverine（MDC） staining. Hep G2 cells were treated with 20 μmol/L Res and 20 μmol/L Res ＋ 5 mmol/L 3-methyladenine（3-MA） for 24 h, and determined for the expressions of Beclin 1,LC3 and P62 by Western blot. Meanwhile, the expressions of PI3 K, p-Akt（Akt） and p-m TOR（m ROR） in PI3 K/Akt/m TOR pathway were determined by Western blot. Results Res inhibited the proliferation of Hep G2 cells in a dose-and time-dependent manner, while promoted the formation of autophgosome. Western blot showed that Res increased the ratio of LC3Ⅱ/LC3Ⅰand the expression of Beclin 1, and decreased the expression of p62. However, after treatment with 3-methyladenine（3-MA）, both the ratio of LC3Ⅱ/LC3Ⅰ and the expression of Beclin 1 decreased, while the expression of p62 increased. Meanwhile, Res also significantly decreased the expressions of PI3 K, p Akt and p-m TOR in the cells.Conclusion Res may induce autophagy to inhibit the proliferation of Hep G2 cells, of which the mechanism may be associated with the inhibitory effect of Res on PI3 K/Akt/m TOR pathway, the up-regulation of expressions of Beclin 1 and LC3 and the down-regulation of expression of p62.